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Pharmacological targeting of Hedgehog (HH)/GLI has proven effective for certain blood, brain and skin cancers including basal cell carcinoma (BCC). However, limited response rates and the development of drug resistance call for improved anti-HH therapies that take into account synergistic crosstalk mechanisms and immune evasion strategies.In previous work, we demonstrated that crosstalk of HH/GLI with pro-inflammatory Interleukin-6 (IL6) signaling drives BCC by promoting tumor cell proliferation [1]. In the present study, we screened for possible mechanisms of cancer immune evasion regulated by synergistic HH-IL6 signaling and identified the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a novel transcriptional target co-regulated by HH-IL6 signaling. Analysis of thecis-regulatory region of IDO1 by chromatin-immunoprecipitation revealed co-occupancy of this region by HH- IL6 induced GLI1 and STAT3 transcription factors along with active chromatin marks at the histone level. Elevated expression of IDO1 in human BCC with high-level HH and IL6 signatures supports the clinical relevance of our mechanistic data. Genetic inhibition of GLI1 expression prevented the induction of IDO1 expression in response to IL6/STAT3 and IFNγ/STAT1 signaling in human melanoma cells. Pharmacological targeting of HH signaling at the level of GLI proteins interfered with IDO1 expression and consequently prevented the production of the immunosuppressive metabolite kynurenine generated by active IDO1 from tryptophan. Further, inhibition of GLI1 enhanced the efficacy of the selective IDO1 inhibitor epacadostat. Of note, inhibition of HH/GLI signaling in melanoma cells not only reduced IDO1 expression but also interfered with the repression of T cell activation by attenuating IDO1/kynurenine-mediated immunosuppression. These data identify the immunosuppressive IDO1-kynurenine pathway as a novel pro-tumorigenic effector of oncogenic HH-IL6 and GLI-STAT cooperation. Our data suggest simultaneous pharmacological targeting of the HH/GLI, JAK/STAT and IDO1- kynurenine axis as rational combination therapy in skin cancers.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin-6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis-regulatory sequences by co-binding of GLI and STAT3 to common HH-IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI-driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH-IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients.

Metastatic melanoma is hallmarked by its ability to switch oncogenic MITF expression. Here we tested the impact of STAT3 on melanoma onset and progression in association with MITF expression levels. We established a mouse melanoma model for deleting Stat3 specifically in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a deficient background. Mice with tissue specific Stat3 deletion showed an early onset of disease, but displayed significantly diminished lung metastases. Whole genome expression profiling also revealed a reduced invasion phenotype, which was functionally confirmed in 3D melanoma model systems. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in up-regulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3 induced CEBPa/b expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that STAT3 enabled CEBPa/b binding to the MITF enhancer region thereby silencing it. We conclude that STAT3 is a metastasis driver in melanoma able to antagonize the MITF oncogene via direct induction of CEBP family member transcription facilitating RAS-RAF-driven melanoma metastasis.List of AbbreviationsATAC-seq, Assay for Transposase-Accessible Chromatin using sequencing; CEBP, CAAT Box Enhancer Binding Protein; CRE, Cre recombinase; EGF, Epidermal Growth Factor; GEO, Gene Expression Omnibus; GSEA, Gene Set Enrichment Analysis; HSC70, Heat Shock 70 kDa protein; IHC, Immunohistochemistry; IL-6, Interleukin-6; JAK, Janus Kinase; MITF, Microphthalmia-Associated Transcription Factor; NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice; OSM, Oncostatin M; PDGF, Platelet-Derived Growth Factor; pS, phosphoserine; pY, phosphotyrosine; RAS, Rat Sarcoma; RAF, Rapidly Accelerated Fibrosarcoma; RTK, Receptor Tyrosine Kinase; RT-PCR, Reverse Transcription Polymerase Chain Reaction; S100b, Calcium Binding Protein S100 beta; shRNA, short hairpin RNA; SOX10, Sex Determining Region Y-10; STAT, Signal Transducer and Activator of Transcription; TCGA, The Cancer Genome Atlas; TMA, Tissue Micro Array

Treatment of acute myeloid leukemia (AML), an aggressive and heterogeneous hematological malignancy, remains a challenge. Despite advances in our understanding of the complex genetics and biology of AML pathophysiology, these findings have been translated to the clinic with only limited success, and poor outcomes persist for the majority of patients. Thus, novel treatment strategies are clearly needed for achieving deeper and prolonged remissions and for avoiding the development of resistance. Due to its profound role in (cancer) stem cell biology and differentiation, the Hedgehog (HH)/Glioma-associated Oncogene Homolog (GLI) signaling pathway may be an attractive novel therapeutic target in AML. In this review, we aim to provide a critical and concise overview of the currently known potential and challenges of HH/GLI targeting. We describe the biological role of the HH/GLI pathway in AML pathophysiology. We specifically focus on ways of targeting non-canonical HH/GLI signaling in AML, particularly in combination with standard treatment regimens, which may overcome some hurdles observed with approved HH pathway inhibitors in solid tumors.